We used a simple and efficient method to construct the bicistronic eukaryotic expression vector pIRES2-VEGF165-NT-3. The neurotrophin-3 (NT-3) gene was obtained from the genomic DNA of human peripheral blood mononuclear cells by polymerase chain reaction. The NT-3 cDNA fragment was cloned into the pIRES2-VEGF165-EGFP vector in place of enhanced green fluorescent protein (EGFP) to create the plasmid pIRES2-VEGF165-NT-3. Next, pIRES2-VEGF165-NT-3 was transfected into HEK293 cells, and reverse transcription-polymerase chain reaction and Western blotting were used to test co-expression of the double genes. The vascular endothelial growth factor 165 (VEGF165) and NT-3 genes were cloned; DNA sequencing analysis demonstrated that the VEGF165 and NT-3 sequences were the same as those recorded in GenBank. Restriction analysis indicated that the VEGF165 and NT-3 genes were correctly inserted into the expression vector pIRES2-EGFP. The double gene was expressed at both the mRNA and protein levels. The VEGF165 and NT-3 co-expression plasmid was successfully constructed, providing a novel expression system for further study of the functions of the VEGF165 and NT-3 genes.